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J Biol Chem. 2019 Aug 9;294(32):12146-12156. doi: 10.1074/jbc.RA119.007766. Epub 2019 Jun 21.

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay.

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Yale Center for Molecular Discovery, West Haven, Connecticut 06516.
Basic Science Section, Department of Medicine, National Jewish Health, Denver, Colorado 80206.
Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, Connecticut 06520.
Departments of Ophthalmology and Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
Department of Microbiology, College of Arts and Sciences, University of Tennessee, Knoxville, Tennessee 37996.
Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, Connecticut 06520


Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 μm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 μg/ml without ethanolamine and an MIC50 of 75 and 60 μg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.


Candida albicans; Plasmodium falciparum; Plasmodium knowlesi; antimicrobial agent; enzyme; high-throughput screening (HTS); parasitic disease; phosphatidylethanolamine; phosphatidylserine; phosphatidylserine decarboxylase; phospholipids; plasmodium

[Available on 2020-08-09]

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